Active form of pyridoxal phosphate in glycogen phosphorylase. Phosphorus-31 nuclear magentic resonance investigation.
The substrate analogue alpha-D-glucopyranosyl cyclic 1,2-phosphate has been confirmed to be a good competitive inhibitor of glycogen phosphorylases a and b isolated from rabbit muscle. Effects of tertiary and quaternary structure of the enzyme have been shown to be similar to those induced by the substrate glucose 1-phosphate and different from those of the coincidently binding inhibitor glucose. This information was obtained from study of the ultracentrifugation patterns of the enzyme-inhibitor complex and by determination of its effect on the binding constant for the activator AMP. 31P NMR investigation of the binding of this inhibitor to the enzyme has demonstrated that it both tightens the binding of the nucleotide activator and shifts the resonance of the phosphate group of the pyridoxal phosphate residue to a broad signal around 0 ppm. This situation is further reinforced in the presence of the second substrate, maltopentaose, giving a fully potentiated, but inactive, enzyme-substrate complex. This has not been studied previously by 31 P NMR. The active form of the pyridoxal phosphate (PLP), in the presence of substrates or their analogues, is not therefore a mobile dianionic phosphate as has been previously proposed. It may represent a tightly bound and constrained dianionic phosphate or possible a protonated phosphate in intermediate exchange. The implications of this finding are discussed.
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010762:Phosphorylase a / Q000378:metabolism; D010763:Phosphorylase b / Q000378:metabolism; D006005:Phosphorylases / Q000378:metabolism*; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000032:analysis*; D011817:Rabbits; D013873:Thionucleotides / Q000494:pharmacology
D013873:Thionucleotides; C014308:adenosine 5'-phosphorothioate; D000249:Adenosine Monophosphate; D011732:Pyridoxal Phosphate; D010762:Phosphorylase a; D010763:Phosphorylase b; D006005:Phosphorylases